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Deep breathing exercises are the second most used complementary health approach in the United States. Two heart rate variability (HRV) parameters, the root mean square of successive differences (RMSSD) and the respiratory sinus arrhythmia (RSA), are used to assess parasympathetic reactivity to deep breathing, but they are often not in agreement. Our purpose was to determine the cause of the disagreement. We investigated HRV parameters in 38 subjects during baseline, deep breathing, and recovery. Here we show that RMSSD as a measure of parasympathetic reactivity is unreliable; it does not reflect the increase in HRV during deep breathing as determined by RSA. We observed a decrease in RMSSD values despite a marked increase in HRV as determined by RSA and the standard deviation of normal heartbeat interval (SDNN) in healthy subjects and patients with functional bowel disorders. We show that RSA captures all aspects of HRV, whereas successive differences in heart rate intervals are only a small part of HRV, with decreasing variability during deep breathing in most subjects. We present a new measure of calculating RSA during deep breathing that may become an essential tool for researchers and clinicians. We also provide a unique visualization of the increased heart rate variability during deep breathing. Hence, RMSSD cannot be used to assess parasympathetic reactivity during deep breathing; using RSA is recommended. The use of RMSSD in previous influential studies may have led to erroneous conclusions about parasympathetic reactivity during deep breathing. Its continued use may undervalue the effects of the autonomic nervous system in slow deep breathing. [ABSTRACT FROM AUTHOR]